Antibodies or antigen-binding fragments thereof that bind to mumps virus in targeted manner

Abstract

The invention relates to a method for preparing an antibody or an antigen-binding fragment of the antibody in targeted binding with mumps virus, and a series of neutralizing monoclonal antibodies or antigen-binding fragments of the neutralizing monoclonal antibodies, which have specificity to mumps virus and are bound with high affinity, prepared by the method. The antibody disclosed by the invention has high efficacy and broad spectrum for neutralizing mumps virus infection, and has wide application prospects in diagnosis, prevention and treatment of mumps diseases.

Description

Technical Field

[0001] The present disclosure belongs to the field of biotechnology, and particularly relates to a method for preparing an antibody or an antigen-binding fragment thereof that targets and binds to mumps virus, and the antibody or the antigen-binding fragment thereof obtained by the method. Background Art

[0002] Mumps is caused by the mumps virus (MuV), which is a highly contagious disease. It is usually transmitted to humans through respiratory droplets or secretions through the respiratory tract or oral route, and can cause typical symptoms such as unilateral or bilateral salivary gland swelling. In addition, it can cause inflammation of the testicles, ovaries, pancreas or meninges, and lead to complications such as infertility or deafness. After infection, the mumps virus first invades the oral mucosa and nasal mucosa, proliferates in large quantities in the epithelial tissue, and then enters the blood circulation (first viremia). It then involves the parotid gland and some tissues through the blood flow, and proliferates therein and enters the blood circulation again (second viremia). The replication of the virus in the parotid gland leads to perivascular and interstitial mononuclear cell infiltration, hemorrhage, edema, and necrosis of alveolar and epithelial duct cells. Mumps is a self-limiting disease. There is no specific treatment for the infection. It is symptomatic treatment, mainly focusing on prevention.

[0003] Mumps is a preventable disease. The most widely used mumps vaccine strain in the world is the Jeryl Lynn strain. For more than 40 years, the Jeryl Lynn strain has been the only mumps vaccine used in the United States, and more than 500 million doses have been distributed worldwide. The virus is passaged in embryonated eggs and then cultured in chicken embryo cells to prepare the vaccine.

[0004] my country has been developing mumps vaccines since the 1970s and has been using mumps monovalent vaccines since the 1990s. In 2008, vaccines containing mumps components were included in the national immunization program, and children aged 18 to 24 months were routinely vaccinated with one dose of MMR. Since June 2020, China has begun to implement a two-dose MMR routine immunization program, with one dose each at 8 and 18 months of age. At present, the mumps component of domestically produced MMR uses the S79 strain. The long-term protection against mumps after one dose of MMR is not ideal, and the protection effect of two doses of MMR is significantly better than that of one dose. Since the use of mumps vaccines, although the number of mumps cases has decreased significantly, epidemic mumps occurs every year at home and abroad. According to the mumps outbreaks reported in the literature, mumps infections have occurred many times in China; the results uploaded from the NCBI database show that the mumps strains in my country have the F genotype and the G genotype, mainly the F type, and occasionally the G genotype strains in many places.

[0005] On the one hand, the outbreak of mumps may be due to the weakening of the immune persistence of vaccination over time; on the other hand, another possible factor in the outbreak of mumps is strain variation. The main MuV prevalent in my country is the F genotype, and the most commonly detected MuV in the world is the G genotype. The antigenic differences between the currently prevalent wild-type virus and the vaccine virus may reduce the recognition of vaccine-induced antibodies. Although vaccine-induced antibodies can neutralize G genotype strains, the neutralization level is lower than that of the vaccine strain against this genotype.

[0006] Therefore, the research and development of new mumps vaccines and specific therapeutic drugs are of great significance for the prevention and control of mumps epidemics. Summary of the invention

[0007] In order to solve the above problems, the present disclosure provides a neutralizing monoclonal antibody or an antigen-binding fragment thereof that is specific to mumps virus and binds with high affinity, as well as a preparation method for producing such an antibody. The present disclosure successfully obtains multiple monoclonal antibodies with high neutralizing activity, and has a high cross-neutralization level for genotype F, genotype A, and genotype G. The development of the disclosed antibodies and preparation methods thereof has broad application prospects in the diagnosis, prevention, and treatment of mumps diseases.

[0008] According to a first aspect of the present disclosure, a method for preparing an antibody or an antigen-binding fragment thereof that targets and binds to mumps virus is provided, the method comprising immunizing an animal with a whole virus of mumps virus to obtain the antibody or the antigen-binding fragment thereof.

[0009] In some embodiments, the whole mumps virus comprises a whole virus of the F genotype mumps virus.

[0010] In some embodiments, the method further comprises using an extracellular domain fragment containing hemagglutinin-neuraminidase protein (HN protein) of mumps virus as a target protein to screen the antibody or antigen-binding fragment thereof.

[0011] In some embodiments, the extracellular domain fragment of the HN protein has amino acids 106 to 582 corresponding to the HN protein of the mumps virus genotype F strain.

[0012] In some embodiments, the extracellular domain fragment of the HN protein has the amino acid sequence of SEQ ID NO:39, or an amino acid sequence having at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0013] In some embodiments, a secretory expression signal peptide is connected to the N-terminus of the extracellular domain fragment of the HN protein.

[0014] In some embodiments, the secretory expression signal peptide is Human IgG2H. Preferably, Human IgG2H has the amino acid sequence of SEQ ID NO:39.

[0015] In some embodiments, the animal comprises a mouse, a rat, a rabbit, a guinea pig, a sheep, or a goat.

[0016] In some embodiments, the method comprises preparing the antibody or antigen-binding fragment thereof using phage display method, hybridoma antibody preparation method and / or single B cell screening method.

[0017] In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a recombinantly expressed antibody.

[0018] According to another aspect of the present disclosure, provided is an antibody or an antigen-binding fragment thereof obtained by the method of the present disclosure.

[0019] According to another aspect of the present disclosure, there is provided an antibody or an antigen-binding fragment thereof that targets and binds to mumps virus, wherein the antibody or the antigen-binding fragment thereof comprises:

[0020] (1) The following three heavy chain variable region complementarity determining regions (HCDRs):

[0021] HCDR1 having the amino acid sequence of HCDR1 contained in the heavy chain variable region as shown in any one of SEQ ID NO: 1, 3, 5, 7 or 10, or an amino acid sequence having one or more amino acids substituted, deleted or added compared to the amino acid sequence of HCDR1 contained in the heavy chain variable region;

[0022] HCDR2 having the amino acid sequence of HCDR2 contained in the heavy chain variable region as shown in any one of SEQ ID NO: 1, 3, 5, 7 or 10, or an amino acid sequence having one or more amino acids substituted, deleted or added compared to the amino acid sequence of HCDR2 contained in the heavy chain variable region;

[0023] HCDR3 having the amino acid sequence of HCDR3 contained in the heavy chain variable region as shown in any one of SEQ ID NO: 1, 3, 5, 7 or 10, or an amino acid sequence having one or more amino acids substituted, deleted or added compared to the amino acid sequence of HCDR3 contained in the heavy chain variable region; and / or

[0024] (2) The following three light chain variable region complementarity determining regions (LCDR):

[0025] LCDR1 having an amino acid sequence of LCDR1 contained in a light chain variable region as shown in any one of SEQ ID NOs: 2, 4, 6, 8, 9 or 11, or an amino acid sequence having one or more amino acids substituted, deleted or added compared to the amino acid sequence of LCDR1 contained in the light chain variable region;

[0026] LCDR2 having an amino acid sequence of LCDR2 contained in a light chain variable region as shown in any one of SEQ ID NOs: 2, 4, 6, 8, 9 or 11, or an amino acid sequence having one or more amino acids substituted, deleted or added compared to the amino acid sequence of LCDR2 contained in the light chain variable region;

[0027] LCDR3, which has the amino acid sequence of LCDR3 contained in the light chain variable region as shown in any one of SEQ ID NO: 2, 4, 6, 8, 9 or 11, or an amino acid sequence having one or several amino acids substituted, deleted or added compared to the amino acid sequence of LCDR3 contained in the light chain variable region.

[0028] In some embodiments, the antibody or antigen-binding fragment thereof comprises: HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region as shown in SEQ ID NO:1, and LCDR1, LCDR2 and LCDR3 contained in the light chain variable region as shown in SEQ ID NO:2.

[0029] In some embodiments, the antibody or antigen-binding fragment thereof comprises: HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region as shown in SEQ ID NO:3, and LCDR1, LCDR2 and LCDR3 contained in the light chain variable region as shown in SEQ ID NO:4.

[0030] In some embodiments, the antibody or antigen-binding fragment thereof comprises: HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region as shown in SEQ ID NO:5, and LCDR1, LCDR2 and LCDR3 contained in the light chain variable region as shown in SEQ ID NO:6.

[0031] In some embodiments, the antibody or antigen-binding fragment thereof comprises: HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region as shown in SEQ ID NO:7, and LCDR1, LCDR2 and LCDR3 contained in the light chain variable region as shown in SEQ ID NO:8.

[0032] In some embodiments, the antibody or antigen-binding fragment thereof comprises: HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region as shown in SEQ ID NO:3, and LCDR1, LCDR2 and LCDR3 contained in the light chain variable region as shown in SEQ ID NO:9.

[0033] In some embodiments, the antibody or antigen-binding fragment thereof comprises: HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region as shown in SEQ ID NO:10, and LCDR1, LCDR2 and LCDR3 contained in the light chain variable region as shown in SEQ ID NO:11.

[0034] In some embodiments, the antibody or antigen-binding fragment thereof comprises: the HCDR1-3 and / or the LCDR1-3 are defined by the Kabat numbering system, the Chothia numbering system, the IMGT numbering system, the Martin numbering system, the Contact numbering system, the Honegger numbering system, the Gelfand numbering system or a combination thereof.

[0035] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment thereof includes: a HCDR1 as shown in any one of SEQ ID NOs: 12, 22 or 33; a HCDR2 as shown in any one of SEQ ID NOs: 13, 18, 23 or 34; and / or a HCDR3 as shown in any one of SEQ ID NOs: 14, 24, 28 or 35.

[0036] In some embodiments, the light chain variable region of the antibody or antigen-binding fragment thereof includes: LCDR1 as shown in any one of SEQ ID NO:15, 19, 25, 29, 30 or 36; and / or LCDR2 as shown in any one of SEQ ID NO:16, 20, 26, 31 or 37; and / or LCDR3 as shown in any one of SEQ ID NO:17, 21, 27, 32 or 38.

[0037] In some embodiments, the heavy chain variable region of the antibody or its antigen-binding fragment comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, respectively, and the light chain variable region of the antibody or its antigen-binding fragment comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:17, respectively.

[0038] In some embodiments, the heavy chain variable region of the antibody or its antigen-binding fragment comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:12, SEQ ID NO:18 and SEQ ID NO:14, respectively, and the light chain variable region of the antibody or its antigen-binding fragment comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21, respectively.

[0039] In some embodiments, the heavy chain variable region of the antibody or its antigen-binding fragment comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24, respectively, and the light chain variable region of the antibody or its antigen-binding fragment comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:27, respectively.

[0040] In some embodiments, the heavy chain variable region of the antibody or its antigen-binding fragment comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:28, respectively, and the light chain variable region of the antibody or its antigen-binding fragment comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:29, SEQ ID NO:20 and SEQ ID NO:21, respectively.

[0041] In some embodiments, the heavy chain variable region of the antibody or its antigen-binding fragment comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:12, SEQ ID NO:18 and SEQ ID NO:14, respectively, and the light chain variable region of the antibody or its antigen-binding fragment comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:32, respectively.

[0042] In some embodiments, the heavy chain variable region of the antibody or its antigen-binding fragment comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:33, SEQ ID NO:34 and SEQ ID NO:35, respectively, and the light chain variable region of the antibody or its antigen-binding fragment comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38, respectively.

[0043] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NO: 1, 3, 5, 7 or 10, an amino acid sequence having one or several amino acids substituted, deleted or added thereto, or an amino acid sequence having at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0044] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region having an amino acid sequence as shown in any one of SEQ ID NO: 2, 4, 6, 8, 9 or 11, an amino acid sequence having one or several amino acids substituted, deleted or added thereto, or an amino acid sequence having at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0045] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region as shown in SEQ ID NO:1 and a light chain variable region as shown in SEQ ID NO:2.

[0046] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region as shown in SEQ ID NO:3 and a light chain variable region as shown in SEQ ID NO:4.

[0047] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region as shown in SEQ ID NO:5 and a light chain variable region as shown in SEQ ID NO:6.

[0048] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region as shown in SEQ ID NO:7 and a light chain variable region as shown in SEQ ID NO:8.

[0049] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region as shown in SEQ ID NO:3 and a light chain variable region as shown in SEQ ID NO:9.

[0050] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region as shown in SEQ ID NO:10 and a light chain variable region as shown in SEQ ID NO:11.

[0051] In some embodiments, the antibody is of the IgA, IgD, IgE, IgG or IgM type.

[0052] In some embodiments, the antibody or antigen-binding fragment thereof is a scFv, Fab, Fab', (Fab')2, Fv fragment, dsFv, diabody, bispecific antibody and multispecific antibody.

[0053] In some embodiments, the antibody or antigen-binding fragment thereof is a chimeric antibody or a humanized antibody.

[0054] In some embodiments, the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region and / or a light chain constant region.

[0055] In some embodiments, the heavy chain constant region is selected from IgG1, IgG2, IgG3 or IgG4 heavy chain constant region. In some embodiments, the heavy chain constant region is selected from hIgG1, hIgG2, hIgG3 or hIgG4 heavy chain constant region.

[0056] According to another aspect of the present disclosure, a chimeric antigen receptor is provided, which includes an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain, wherein the extracellular antigen binding domain comprises the antibody or antigen-binding fragment thereof prepared by the method described in the present disclosure, or the antibody or antigen-binding fragment thereof.

[0057] According to yet another aspect of the present disclosure, a modified immune cell is provided, which comprises the chimeric antigen receptor described in the present disclosure.

[0058] According to another aspect of the present disclosure, an isolated nucleic acid is provided, which encodes the antibody or antigen-binding fragment thereof prepared by the method described in the present disclosure, or the antibody or antigen-binding fragment thereof.

[0059] In some embodiments, the isolated nucleic acid is an mRNA molecule.

[0060] According to yet another aspect of the present disclosure, an expression vector is provided, comprising the isolated nucleic acid described in the present disclosure.

[0061] In some embodiments, the expression vector can be a eukaryotic cell expression vector and / or a prokaryotic cell expression vector, such as a retroviral vector, a lentiviral vector, a phage vector, an adenoviral vector, an adeno-associated vector or a herpes simplex vector.

[0062] In some embodiments, the expression vector is present in nanoparticles, liposomes, exosomes, microvesicles, or a gene gun.

[0063] According to another aspect of the present disclosure, a host cell is provided, which comprises the isolated nucleic acid or the expression vector described in the present disclosure.

[0064] In some embodiments, the host cell is a conventional host cell in the art, as long as the expression vector can stably express the carried nucleic acid molecule as the antibody or antigen-binding fragment thereof described in the present disclosure.

[0065] In some embodiments, the host cell is a prokaryotic cell and / or a eukaryotic cell, the prokaryotic cell is preferably an E. coli cell such as TG1, BL21 (expressing a single-chain antibody or a Fab antibody), and the eukaryotic cell is preferably a HEK293 cell or a CHO cell (expressing a full-length IgG antibody). The expression vector is transformed into a host cell to obtain the host cell of the present disclosure. The transformation method is a conventional transformation method in the art, preferably a chemical transformation method, a heat shock method or an electroporation method.

[0066] According to another aspect of the present disclosure, an antibody-drug conjugate is provided, comprising: an antibody or an antigen-binding fragment thereof prepared by the method described in the present disclosure, or the antibody or the antigen-binding fragment thereof; and a ligand.

[0067] In some embodiments, the ligand is selected from the group consisting of a radioisotope, a fluorescent group, and a delivery vehicle.

[0068] According to another aspect of the present disclosure, a pharmaceutical composition is provided, comprising: the antibody or antigen-binding fragment thereof prepared by the method described in the present disclosure, or the antibody or antigen-binding fragment thereof, or the antibody-drug conjugate; and a pharmaceutically acceptable carrier.

[0069] In some embodiments, the pharmaceutically acceptable carrier may be a conventional carrier in the art, and the carrier may be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipient. The pharmaceutical excipient is a conventional pharmaceutical excipient in the art, preferably including a pharmaceutically acceptable excipient, filler or diluent, etc. More preferably, the pharmaceutical composition comprises 0.01 to 99.99% of the antibody or its antigen-binding fragment and / or other small molecule drugs or antibodies or polypeptides, and 0.01 to 99.99% of a pharmaceutical carrier, and the percentage is the mass percentage of the pharmaceutical composition.

[0070] In some embodiments, the route of administration of the pharmaceutical composition can be parenteral, injection or oral administration. The pharmaceutical composition can be prepared in a form suitable for administration, such as solid, semisolid or liquid form, and can be in the form of an aqueous solution, non-aqueous solution or suspension, powder, tablet, capsule, granule, injection or infusion. It can be administered intravascularly, subcutaneously, intraperitoneally, intramuscularly, by inhalation, intranasally, by airway instillation or intrathoracic instillation. The pharmaceutical composition can also be administered in the form of an aerosol or spray, such as nasal administration; or, intrathecally, intramedullary or intraventricular administration, and can also be administered transdermally, percutaneously, topically, enterally, sublingually or rectally. The pharmaceutical composition can be prepared in various dosage forms as needed, and can be administered by a physician according to factors such as the patient's type, age, weight and general disease condition, and the mode of administration to determine the dosage that is beneficial to the patient.

[0071] In some embodiments, the antibody or antigen-binding fragment thereof and other active ingredients in the pharmaceutical composition can be administered simultaneously or sequentially.

[0072] According to another aspect of the present disclosure, there is provided an antibody or antigen-binding fragment thereof prepared by the method described in the present disclosure, or the antibody or antigen-binding fragment thereof, or the isolated nucleic acid, or the antibody-drug conjugate, or the pharmaceutical composition for preparing a drug for preventing, treating and / or diagnosing a disease caused by mumps virus, or for detecting mumps virus or HN protein of mumps virus in vitro.

[0073] According to another aspect of the present disclosure, a kit for in vitro detection of mumps virus or HN protein of mumps virus is provided, characterized in that it comprises the antibody or antigen-binding fragment thereof prepared by the method described in the present disclosure, or the antibody or antigen-binding fragment thereof, or the isolated nucleic acid.

[0074] In some embodiments, the antibody or antigen-binding fragment thereof is labeled with a marker. In some embodiments, the marker is selected from an enzyme, a chemiluminescent group, and an isotope group.

[0075] According to another aspect of the present disclosure, a method for preventing and / or treating mumps virus infection is provided, comprising administering to a subject an effective amount of the antibody or antigen-binding fragment thereof prepared by the method described in the present disclosure, the antibody or antigen-binding fragment thereof, the chimeric antigen receptor, the antibody-drug conjugate or the pharmaceutical composition.

[0076] According to another aspect of the present disclosure, a method for detecting mumps virus or HN protein of mumps virus in a sample is provided, the method comprising the steps of detecting mumps virus or HN protein of mumps virus in a sample using the antibody or antigen-binding fragment thereof prepared by the method described in the present disclosure, or the antibody or antigen-binding fragment thereof,

[0077] Preferably, the sample is whole blood, red blood cell concentrate, platelet concentrate, white blood cell concentrate, tissue, bone marrow aspirate, plasma, serum, cerebrospinal fluid, feces, urine, cultured cells, saliva, oral secretions and / or nasal secretions.

[0078] Compared with the prior art, the present invention has the following technical effects:

[0079] (1) The present disclosure uses the domestic mumps virus epidemic genotype as the research target for whole virus immunization, and uses the recombinant expression strategy to select the neutralizing antigen HN protein as the screening target, successfully obtaining multiple strains of specific binding monoclonal antibodies that can be used for mumps virus detection and analysis. The animal immunization of the present disclosure includes but is not limited to mice, rats, rabbits, guinea pigs, goats, etc.

[0080] (2) The present disclosure utilizes phage display antibody preparation technology to improve antibody diversity, successfully screens and obtains monoclonal antibodies with broad cross-neutralization levels against genotypes F, A, and G. The obtained antibodies have good specificity, high antigen recognition target, and low non-specificity. The antibody preparation technology disclosed in the present disclosure includes, but is not limited to, phage display technology, and also includes hybridoma, single B cell screening technology, etc.

[0081] (3) The specific antibodies obtained by screening in the present disclosure are monoclonal antibodies with high neutralizing activity and can inhibit mumps infection; at the same time, the monoclonal antibodies disclosed in the present disclosure are recombinantly expressed antibodies, which can be scaled up and produced, and have good commercial value. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] Figure 1 The figure shows the SDS-PAGE results after purification of the recombinant expressed HN protein, where NR is a non-reducing gel and R is a reducing gel.

[0083] Figure 2 The non-reducing gel results of SDS-PAGE after purification of the recombinant expressed antibody are shown, wherein lanes 1, 2, 3, 4, 5, and 6 are mumps monoclonal antibodies R001, R003, R008, R046, R050, and R066, respectively, and lane M is a marker protein.

[0084] Figure 3The reducing gel results of SDS-PAGE after purification of the recombinant expressed antibody are shown, wherein lanes 1, 2, 3, 4, 5, and 6 are mumps monoclonal antibodies R001, R003, R008, R046, R050, and R066, respectively, and lane M is a marker protein.

[0085] Figure 4 The results show the binding specificity of the recombinantly expressed antibody to the HN protein.

[0086] Figure 5 The results show the binding specificity of the recombinantly expressed antibody to the inactivated mumps F genotype wild strain. DETAILED DESCRIPTION

[0087] In order to make the purpose, technical scheme and advantages of the present disclosure more clear, the present disclosure is further described in detail below in conjunction with the accompanying drawings and embodiments. The specific embodiments described herein are only used to explain the present disclosure and are not intended to constitute any limitation to the present disclosure. In addition, in the following description, the description of well-known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0088] Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly used in the field to which this disclosure belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0089] Unless the context clearly dictates otherwise, the expressions "a", "an" and "an" as used herein include plural references. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

[0090] As used herein, the term "about" refers to a range of ±20% of the value that follows. In some embodiments, the term "about" refers to a range of ±10% of the value that follows. In some embodiments, the term "about" refers to a range of ±5% of the value that follows.

[0091] Mumps virus (MuV) belongs to the Paramyxoviride family and the genus Mumpsvirus. It is the pathogen that causes epidemic mumps (abbreviated as mumps). The MuV genome is a non-segmented single-stranded negative-strand ribonucleic acid (RNA) containing 15,384 nucleotides. The whole genome sequence encodes seven viral protein genes in the order of 3'-nucleocapsid protein (N)-phosphorylated protein (P)-matrix protein (M)-fusion protein (F)-small hydrophobic protein (SH)-hemagglutinin / neuraminidase protein (HN)-polymerase protein (L)-5'. Among them, the SH gene has the largest degree of variation and is the basis for mumps genotyping. Based on the differences in SH gene nucleotides, 12 MuV genotypes have been discovered worldwide, named A to N genotypes (A, B, C, D, F, G, H, I, J, K, L, N). There are multiple wild-type MuV strains circulating around the world, of which C, D and H genotypes are mainly distributed in the Western Hemisphere, B, F, I and L genotypes are mainly distributed in Asia, and G genotype has been detected in most countries or regions around the world. The main mumps virus currently prevalent in my country is the F genotype, and the G genotype has appeared in some local areas and is gradually expanding.

[0092] Mumps virus invades host cells mainly through surface HN protein and F protein. MuV-HN is a type II membrane protein, which consists of an N-terminal cytoplasmic tail, a transmembrane region, a stalk, and a C-terminal receptor binding domain. The receptor binding domain of the MuV-HN monomer is a six-leaf β-helix fold, which has a specific affinity for sugar chains containing α-2,3-chain sialic acid. During the infection process, the MuV-HN protein binds to sialic acid on the cell surface to infect the upper respiratory tract. After receptor binding, the multimerization of the MuV-HN head domain may affect the structural changes of the MuV-HN stalk domain and stimulate the structural changes of the adjacent F protein; the F protein is a viral membrane fusion protein. The host endopeptide (furin) recognizes the RXL / RR sequence of the F protein and cleaves it into an active protein, which changes from pre-fusion to post-fusion. This structural change of the F protein provides the ability of the viral envelope to fuse with the cell membrane, induces pH-independent fusion of the viral envelope with the host plasma membrane, helps the virus penetrate the cell surface, and releases the viral genomic RNA into the cytoplasm for transcription and replication. Once the virus enters the polarized epithelial cells of the respiratory tract, it can enhance the invasion of mumps virus into its neighboring cells. MuV is secreted from the epithelial cells, resulting in viral growth in the glandular epithelium and shedding in the saliva. MuV can be spread systemically in the human body, causing viremia early in the infection.

[0093] The term "antibody" of the present disclosure encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies or trispecific antibodies), single-chain molecules, and antibody fragments, as long as the desired antigen-binding activity is exhibited.

[0094] The term "monoclonal antibody" disclosed herein refers to an antibody obtained from a substantially homogeneous antibody group, i.e., except for variant antibodies that may be present in trace amounts (e.g., containing naturally occurring mutations or produced during the production of monoclonal antibody preparations, usually present in small amounts), each antibody contained in the antibody group is identical and / or binds to the same epitope. Different from polyclonal antibody preparations that typically include different antibodies directed against different antigenic determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on the antigen.

[0095] The terms "antibody or its antigen-binding fragment" and "antibody" of the present disclosure are used interchangeably herein to refer to antibodies that are substantially similar to natural antibody structures. "Native antibody" refers to naturally occurring immunoglobulin molecules. For example, natural IgG class antibodies are heterotetrameric glycoproteins of about 150,000 daltons, consisting of two light chains and two heavy chains bonded by disulfide bonds. From N-terminal to C-terminal, each heavy chain has a variable region (VH) (also referred to as a variable heavy chain domain or a heavy chain variable domain) and three constant domains (CH1, CH2 and CH3) (also referred to as a heavy chain constant region). From N-terminal to C-terminal, each light chain has a variable region (VL) (also referred to as a variable light chain domain or a light chain variable domain) and a light chain constant domain (CL) (also referred to as a light chain constant region). The heavy chain of an antibody can be one of five types, α (IgA), δ (IgD), ε (IgE), γ (IgG) or μ (IgM), which can be further divided into subtypes, such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The light chain of an antibody can be one of two types, κ light chain and λ light chain, based on the amino acid sequence of its constant domain.

[0096] Within the light chain and heavy chain, the variable region and the constant region are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2 and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant region of the antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0097] The term "variable region" or "variable domain" of the present disclosure refers to the domain of the antibody heavy chain or light chain that participates in the binding of the antigen-binding molecule to the antigen. The variable domains of the heavy chain and light chain of natural antibodies (VH and VL, respectively) generally have similar structures, wherein each domain comprises four conserved framework regions (FR) and three hypervariable regions (HVR). A single VH or VL domain may be sufficient to confer antigen-binding specificity.

[0098] The term "variable" disclosed herein refers to certain segments of the variable domain that are generally different in sequence between antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its specific antigen. However, variability is not evenly distributed throughout the variable domain. Instead, it is concentrated in three segments called hypervariable regions (HVRs) within the light chain and heavy chain variable domains. The more highly conserved parts of the variable domain are called framework regions (FRs). The variable domains of the natural heavy and light chains each contain four FR regions, most of which adopt a β-folded configuration, connected by three HVRs, which form a loop connection and form a part of the β-folded structure in some cases. The HVRs in each chain are closely held together by the FR region, and together with the HVRs of other chains, contribute to the formation of the antigen binding site of the antibody (see Kabat et al., Sequences of Immunological Interest, 5th edition, National Institute of Health, Bethesda, MD (1991)). The constant domain is not directly involved in the binding of the antibody to the antigen, and has other effector functions, such as participating in the antibody-dependent cellular toxicity of the antibody.

[0099] The term "hypervariable region" or "HVR" of the present disclosure refers to a region in the variable domain region of an antibody that is highly variable in sequence and / or forms a structurally defined loop ("hypervariable loop"). Typically, a natural four-chain antibody comprises six HVRs: three present in VH (H1, H2, H3) and three present in VL (L1, L2, L3). HVRs typically comprise amino acid residues from hypervariable loops and / or from "complementarity determining regions (CDRs)", which have the highest sequence variability and / or are involved in antigen recognition.

[0100] As used herein, the term "complementarity determining region (CDR)" is the amino acid residue in the variable region of an antibody that is responsible for antigen binding. The precise boundaries of these amino acid residues can be defined according to various numbering systems known in the art, such as the Kabat numbering system, the Chothia numbering system, the IMGT numbering system, the Martin numbering system, the Contact numbering system, the Honegger numbering system, the Gelfand numbering system, or a combination thereof. For a given antibody, a person skilled in the art will easily identify the CDRs defined by each numbering system. The corresponding relationship between different numbering systems is well known to those skilled in the art, and some conventionally used software can be used to define HCDR1-3 and LCDR1-3. For example, the Kabat numbering system is an immunoglobulin alignment and numbering system proposed by Elvin A.Kabat (see, for example, Kabat et al., (1991), Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.). The Chothia numbering system is an immunoglobulin numbering system proposed by Chothia et al., which is a classic rule for identifying CDR region boundaries based on the position of the structural loop region (see, for example, Chothia & Lesk, (1987), J. Mol. Biol. 196: 901-917; Chothia et al., (1989), Nature 342: 878-883; Al-Lazikani et al., (1997), JMB 273: 927-948). The IMGT numbering system is a numbering system based on the international ImMunoGeneTics information (IMGT) initiated by Lefranc et al. (see, for example, Lefranc et al., (2003), Dev. Comparat. Immunol. 27: 55-77). The Martin numbering system is a new numbering scheme recommended by Martin et al. for structural alignment of different framework regions with unconventional lengths, which can be completed by the Chothia numbering system corrected by ABnum software. The Contact numbering system defines CDR regions based on available crystal structure data for antibody complexes. The Honegger numbering system (also known as the AHo numbering system) is based on structural alignments of 3D structures of immunoglobulin variable regions covering the observed length variation, allowing the definition of structurally conserved Cα positions and therefore the deduction of appropriate FR region and CDR lengths.The Gelfand numbering system is a relatively complex numbering scheme described by Gelfand et al., in which variable chain sequences are divided into 21 segments called "words", each of these "words" matches a secondary structure element (chain or loop), chains are defined by alphabetical letters (e.g., A, B, C), and loops are defined by two letters, which correspond to adjacent chains (e.g., AB, BC...), this numbering system does not include gaps or missing points, but allows accurate comparison of secondary structures (loops and chains) between aligned sequences.

[0101] The term "framework" or "FR" of the present disclosure refers to the variable domain residues other than the hypervariable region (HVR) residues. The FR of the variable domain is generally composed of the following four FR domains: FR1, FR2, FR3 and FR4. Therefore, HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1 (L1)-FR2-H2 (L2)-FR3-H3 (L3)-FR4.

[0102] The "class" of an antibody refers to the type of constant domain or constant region possessed by the heavy chain of the antibody. There are five classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0103] The term "humanized antibody" of the present disclosure comprises amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody comprises at least one, usually two variable domains, wherein all or substantially all HVRs (e.g., CDRs) correspond to HVRs of non-human antibodies, and all or substantially all FRs correspond to FRs of human antibodies. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. An antibody in a "humanized form," such as a non-human antibody, refers to an antibody that has undergone humanization.

[0104] A "humanized antibody" has an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or human cell or derived from a non-human source utilizing a human antibody library or other human antibody encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.

[0105] The disclosure also relates to amino acid sequence variants, which can be prepared by introducing appropriate modifications into the nucleotide sequence of the encoding molecule or by peptide synthesis. Such modifications include, for example, deletions, insertions and / or substitutions of residues in the antibody amino acid sequence. Any combination of deletions, insertions and substitutions can be performed to obtain a final construct having the desired properties, such as antigen binding activity. Sites for substitution generally include HVR and framework (FR). See the possible substitutions of amino acids in the table below.

[0106] Conservative amino acid substitution

[0107]

[0108]

[0109] The term "polynucleotide" or "nucleic acid" or "nucleotide sequence" of the present disclosure refers to an isolated nucleic acid molecule or construct, such as messenger RNA (mRNA), virally derived RNA or plasmid DNA (pDNA). A polynucleotide may contain conventional phosphodiester bonds or unconventional bonds (e.g., amide bonds, such as those found in peptide nucleic acids (PNA)). The term "nucleic acid molecule" refers to any one or more nucleic acid segments present in a polynucleotide, such as DNA or RNA fragments.

[0110] The term "antibody fragment" or "antigen-binding fragment" of the present disclosure comprises a portion of a complete antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv; diabodies, triabodies, tetrabodies, crossover Fab fragments; linear antibodies; single-chain antibody molecules (e.g., scFv); multispecific antibodies formed from antibody fragments and single domain antibodies (single domain antibodies).

[0111] The term "antigen binding domain" or "antigen binding site" disclosed herein refers to the part of an antigen binding molecule that specifically binds to an antigenic determinant. More specifically, the term "antigen binding domain" refers to a part of an antibody that includes a region that specifically binds to and is complementary to a part or all of an antigen. In the case where the antigen molecule is very large, the antigen binding molecule can only bind to a specific part of the antigen, which is called an epitope. The antigen binding domain can be provided by, for example, one or more variable domains (also referred to as variable regions). Preferably, the antigen binding domain includes an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). In one aspect, the antigen binding domain is capable of binding to its antigen and blocking or partially blocking the function of the antigen.

[0112] The term "antigenic determinant" of the present disclosure is synonymous with "antigen" and "epitope", and refers to a site on a polypeptide macromolecule (e.g., a stretch of continuous amino acids or a conformational configuration consisting of different regions of non-continuous amino acids), to which an antigen-binding portion binds, thereby forming an antigen-binding portion-antigen complex. Antigenic determinants may be present, for example, on the surface of tumor cells, on the surface of microbially infected cells, on the surface of other diseased cells, on the surface of immune cells, in free matter in serum, and / or in the extracellular matrix (ECM). Unless otherwise indicated, the protein used as an antigen in the present disclosure may be any natural form of protein from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The antigen may also be a human protein, or the antigen may be a "full-length", unprocessed protein, as well as any form of protein produced by intracellular processing, or a naturally occurring protein variant, such as a splice variant or an allelic variant.

[0113] The term "specific binding" disclosed herein refers to binding selectivity for an antigen and can be distinguished from unwanted or non-specific binding. The ability of an antigen-binding molecule to bind to a specific antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art (e.g., surface plasmon resonance (SPR) technology and traditional binding assays. In one embodiment, the degree of binding of an antigen-binding molecule to an unrelated protein, as measured by SPR, is less than about 10% of the degree of binding of the antigen-binding molecule to the antigen.

[0114] The term "affinity" or "binding affinity" of the present disclosure refers to the strength of the non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Binding affinity can generally be expressed in terms of a dissociation constant (Kd), which is the ratio of the dissociation rate constant to the association rate constant (Koff and Kon, respectively). Therefore, equivalent affinities can include different rate constants as long as the ratio of the rate constants remains the same. Affinity can be measured by conventional methods known in the art, such as surface plasmon resonance (SPR).

[0115] The term "isolated" nucleic acid molecule or polynucleotide of the present disclosure refers to a nucleic acid molecule, DNA or RNA, that has been separated from its natural environment. In the present disclosure, the recombinant polynucleotide encoding the polypeptide contained in the vector is also isolated. Other examples of isolated polynucleotides include recombinant polynucleotides in heterologous host cells or polynucleotides purified in solution. Isolated polynucleotides include polynucleotide molecules that are usually contained in cells containing the polynucleotide molecules, but the polynucleotide molecules are present in chromosomes or in chromosome positions different from their natural chromosome positions. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the present disclosure, in positive and negative strand forms, and in double-stranded forms. The isolated polynucleotides or nucleic acids of the present disclosure further include such molecules generated synthetically. In addition, the polynucleotides or nucleic acids can be or can include regulatory elements, such as promoters, ribosome binding sites, or transcription terminators.

[0116] The terms "vector" or "expression vector" and "expression construct" disclosed herein can be used interchangeably to refer to a DNA molecule that introduces a specific gene operably linked thereto into a target cell and directs expression. The vector includes a vector that is a self-replicating nucleic acid structure and a vector that is incorporated into the genome of a host cell into which it has been introduced. The expression vector disclosed herein comprises an expression cassette. The expression vector can perform transcription of a large amount of stable mRNA. Once the expression vector is in the target cell, the ribonucleic acid molecule or protein encoded by the gene is generated by the cell transcription and / or translation machinery. The term "expression cassette" disclosed herein refers to a polynucleotide generated by recombinant or synthetic means, having a series of nucleic acid elements that allow a specific nucleic acid to be transcribed in a target cell. The recombinant expression cassette can be introduced into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus or nucleic acid fragment. Typically, the recombinant expression cassette portion of the expression vector includes, in addition to other sequences, a nucleic acid sequence to be transcribed and a promoter.

[0117] The terms "host cell", "host cell line" and "host cell culture" of the present disclosure are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, and also include the progeny of such cells. Host cells include "transformants / transformants" and "transformed cells", including primary transformed cells and progeny derived therefrom. The nucleic acid of the progeny may not be completely identical to that of the parent cell and may contain mutations. Host cells are any type of cells that can be used to produce the antibodies or antigen-binding fragments thereof disclosed herein. Host cells include cultured cells, such as cultured mammalian cells, such as CHO cells, HEK293 cells, BHK cells, NS0 cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells, insect cells and plant cells, and also include cells contained in transgenic animals, transgenic plants or cultured plant or animal tissues.

[0118] The term "antibody drug conjugate" or "ADC" of the present disclosure refers to a binding protein (such as an antibody or an antibody or antigen-binding fragment thereof) chemically linked to one or more chemical drugs. In a preferred embodiment, the ADC includes a binding protein, a drug, and a linker connecting the binding protein to the drug.

[0119] The term "chimeric antigen receptor" or "CAR" disclosed herein refers to a receptor with a desired antigen specificity and a signaling domain to propagate intracellular signals when an antigen binds. For example, T lymphocytes recognize specific antigens via the interaction of a T cell receptor (TCR) with a short peptide presented by a class I or class II major histocompatibility complex (MHC) molecule. For initial activation and clonal expansion, initial T cells rely on antigen presenting cells (APCs) that provide additional co-stimulatory signals. In some embodiments, monocytes and macrophages can be engineered to, for example, express chimeric antigen receptors (CARs). Modified cells can be recruited to the tumor microenvironment, where they are used as powerful immune effectors by infiltrating tumors and killing target cancer cells. CAR may include an antigen binding domain, a transmembrane domain, and an intracellular domain. The antigen binding domain binds to an antigen on a target cell. Examples of cell surface markers that can be used as antigens bound to the antigen binding domains of CAR include those associated with viruses, bacteria, parasitic infections, autoimmune diseases, and cancer cells (e.g., tumor antigens).

[0120] An "effective amount" of a drug of the present disclosure refers to the amount necessary to cause a physiological change in a cell or tissue to which it is administered. An "effective amount" includes an amount sufficient to improve or prevent a symptom or condition of a medical disease. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition to be treated, the patient's overall health, the method, route and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.

[0121] A "therapeutically effective amount" of a drug (e.g., a pharmaceutical composition) refers to an amount necessary to effectively achieve the desired therapeutic or preventive effect at a dosage and dosing interval and time. For example, a therapeutically effective amount of a drug eliminates, alleviates / reduces, delays, minimizes, or prevents the adverse effects of a disease.

[0122] The term "individual" or "subject" of the present disclosure refers to a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats). Specifically, the individual or subject is a human.

[0123] The term "pharmaceutical composition" of the present disclosure refers to a mixture containing one or more antibodies or antibodies or antigen-binding fragments thereof of the present disclosure and other chemical components, such as physiologically / pharmaceutically acceptable carriers or excipients. The purpose of the pharmaceutical composition is to facilitate administration to an organism, facilitate the absorption of the active ingredient, and thus exert biological activity.

[0124] The term "pharmaceutically acceptable carrier" of the present disclosure refers to a component in a pharmaceutical composition other than the active ingredient that is non-toxic to a subject. Pharmaceutically acceptable excipients include, but are not limited to, buffers, stabilizers and / or preservatives.

[0125] Example

[0126] Example 1. Preparation of antibodies

[0127] 1) Design and synthesize the HN protein of MuV F genotype strain.

[0128] Through bioinformatics analysis, the full length of HN protein is 582aa, of which 1-34 is a signal peptide, 35-56 is a transmembrane region, and 57-582 is an extracellular domain. According to the HN protein structure (PDB: 5b2d), it is determined that aa106-582 has a stable structure and function. Therefore, aa106-582 of the HN protein of the F genotype strain was selected as the target sequence for recombinant expression (MEPSKLFTISDNATFAPGPVINAADKKTFRTCFRILVLSVQVVTLILVIVTLGELVRMINDQ GLSNQLSSITNKIKESATMIASAVGVMNQVIHGVTISLPLQIEGNQNQLLSTLATICTNRNQVSNCSTNIPLVNDLRFINGINKFIIEDYATHDFSIGHPLNMPSFIPTATSPNGCTRIPSFSLGKTHWCY THNVINANCKDHTSSNQYVSMGILVQTASGYPMFKTLKIQYLSDGLNRKSCSIATIPDGCAMYCYVSTQLETDDYAGSSPPTQKLTLLFYNDTVIERTISPSGLEGNWATLVPGVGSGIYFENKLIFPAYG GVLPNSTLGVKSAREFFRPVNPYNPCSGPQQDLDQRALRSYFPSYLSNRRVQSAFLVCAWNQILVTNCELVVPSNNQTLMGAEGRVLLINNRLLYYQRSTSWWPYELLYEISFTFTNSGQSSVNMSWIPIYSFTRPGSGNCSGENVCPTACVSGVYLDPWPLTPYSHQSGINRNFYFTGALLNSSTTRVNPTLYVSALNNLKVLAPYGTQGLFASYTTTTCFQDTGDASVYCVYIMELASNIVGEFQILPVLTRLTII, SEQ ID NO:39), Human IgG2H was used as a secretory expression signal peptide (MGWSCIILFLVATATGVHS, SEQ ID NO:40) connected to the N-terminus of the HN protein, the expression system was HEK293 cells, and the target protein was obtained by transient expression using TFI transfection reagent. The expression results are shown in Figure 1 The recombinantly expressed HN protein has uniform properties and high protein purity.

[0129] 2) Immunization of animals with genotype F wild strain.

[0130] The wild-type F virus strain was isolated from a throat swab sample from a child with clinical mumps infection. After Vero cell culture, the virus titer was determined to be about 7.5 lg CCID 50 / mL. Mumps wild strain immunization was carried out in a biosafety level 2 laboratory. Animals (New Zealand white rabbits) were immunized by intraperitoneal injection. The immunization intervals were 0, 7, 14, 28, and 42 days for a total of five times. The sixth or seventh immunization was performed based on the serum titer after immunization.

[0131] 3) Animal serum titer testing.

[0132] The serum titer was detected by indirect ELISA. The solid phase antigen was prepared by coating with recombinantly expressed HN protein. The serum of animals immunized with wild strains was graded and diluted to avoid the former serum as a negative control. Goat anti-rabbit IgG-HRP secondary antibody (Abcam) was used for color development. Serum positive screening was performed based on the absorption value OD450-630 with 2.1×cutoff (negative control), and animals with high absorption values ​​were selected for subsequent antibody preparation. The F genotype HN protein was coated at 1 and 5 μg / mL, and the titer of the peripheral blood of rabbits after five immunizations with the F genotype wild strain was detected. The results are shown in Table 1. The results showed that the rabbit numbered F5-2 had the highest titer level, and the subsequent antibody library preparation could be carried out.

[0133] Table 1

[0134]

[0135]

[0136] 4) Preparation and screening of phage libraries.

[0137] Using HN protein as the screening target, we selected F5-2 rabbits with high IgG binding titer, extracted total RNA from spleen and bone marrow, amplified the VL and VH coding genes of the variable regions of the antibody light chain and heavy chain respectively by RT-PCR, randomly spliced ​​VL and VH into gene fragments scFv single-chain antibodies using Linker, constructed into phage vectors, and electroporated into TG1 competent cells. M13-assisted VCM13 phage was used to infect TG1 Escherichia coli, and PEG was used to precipitate phage particles after infection to obtain phage library. The phage library was obtained by immunization with specific immunogen HN protein, and contained scFv antigen-specific antibodies with high abundance and diversity, with a library capacity of 10 9 cfu, can improve the success rate of screening high-affinity antibodies.

[0138] The phage library is then screened, and the process includes: (a) incubating the phage library with the coated antigen. (b) washing and removing the unbound phage. (c) eluting the phage bound to the antigen. (d) the eluted phage infects the host bacteria, then multiplies and amplifies, and then performs the next round of elution. The above steps are repeated 2-3 times, so that the phage bound to the target antigen is enriched.

[0139] 5) scFv antibody determination, recombinant expression and verification.

[0140] The screened scFv was sequenced and connected to the C-terminus of the Fc functional region of the IgG antibody to construct a full-domain IgG antibody. The light chain and heavy chain of the IgG antibody were constructed into eukaryotic expression vectors respectively. The two plasmids were mixed in a certain ratio in HEK293 cells and then mixed with the transfection reagent TF1 at a ratio of 1:10 (w / v). The mixture was added to the cells to start transfection. After 5-7 days of culture, the cell supernatant was harvested and protein was purified by affinity chromatography. The protein concentration of the process samples and the final protein product in the purification process was calibrated by UV OD280, and the protein purity was calibrated by Coomassie Brilliant Blue SDS-PAGE.

[0141] The results showed that the non-reduced band of the expressed antibody was between 120 and 150 kD. After reduction, clear heavy chain and light chain bands were obtained, indicating that the target antibody was obtained. The purity of the antibody can reach 98% (such as Figure 2 and Figure 3 shown).

[0142] The heavy chain variable region sequences and light chain variable region sequences of each antibody are shown in Table 2 below, and the CDR sequences of each antibody are shown in Table 3 below.

[0143] Table 2

[0144]

[0145]

[0146] Table 3

[0147]

[0148]

[0149] Example 2. Verification of antibody binding specificity

[0150] Solid phase antigens were prepared by coating with the recombinantly expressed HN protein (coating concentration 1 μg / ml) and inactivated mumps F genotype wild strain (coating concentration 20 μg / ml) used in Example 1, and the recombinantly expressed antibody was diluted in series, PBS was used as a negative control, and goat anti-rabbit IgG-HRP secondary antibody (Abcam) was used for color development. According to the absorption value OD450-630, 2.1×cutoff (negative control) was used as the positive judgment standard.

[0151] The results showed that the recombinant expressed antibodies disclosed herein have high binding specificity with HN protein and inactivated mumps F genotype wild strain (eg Figure 4 and Figure 5 shown).

[0152] Example 3. Antibody neutralization titer detection

[0153] The neutralizing antibody titer test adopts the cell neutralization method, and the specific operations include:

[0154] (a) Preparation of cell plates: Vero cells grown to a monolayer were taken, the culture medium was discarded, PBS solution was added to wash the cell surface, and trypsin digestion solution was added to prepare 1.5×10 5 ~2×10 5 Cell suspension of 100 cells / mL was added to each well of a 96-well cell culture plate at 0.1 mL and was available after 30 minutes.

[0155] (b) Mumps virus dilution: Dilute the strain to be neutralized to 1000 CCID using virus diluent (MEM culture medium containing 2.3% 7.5% NaHCO3 and 1% 3% glutamine). 50 / mL;

[0156] (c) Antibody dilution: The recombinant expressed antibody obtained in Example 1 was diluted to 1 mg / mL and then diluted 2-fold. The dilution ratio was adjusted according to the neutralization titer results;

[0157] (d) Virus neutralization: Take 500 μL of each antibody diluted in appropriate multiples into an EP tube, add 500 μL of mumps virus solution, mix well, and place in a 37°C water bath for 60 min. Take 500 μL of virus dilution solution into an EP tube, add 500 μL of 1000 CCID50 / mL virus solution, mix well, and place in a 37°C water bath for 60 min as a positive control;

[0158] (e) Inoculation: Set the test sample, positive control, and cell control and inoculate them into the prepared 96-well cell culture plate, 0.1 mL per well, and inoculate 4 wells;

[0159] (f) Cultivation: The inoculated cell culture plate was placed in a 36.5°C carbon dioxide incubator for 8 days and then taken out and observed under a microscope;

[0160] (g) Potency determination: First, the cell control should show no cytopathic effect, the virus control should show cytopathic effect, the standard should be within the normal calibration range, and the test sample should calculate its half effective concentration (IC) according to the Reed-Muench method. 50 ).

[0161] The calculation formula is: lg IC 50 = the logarithm of the antibody dilution concentration that is higher than or equal to 50% effective + distance ratio × the logarithm of the dilution concentration.

[0162] According to the above method and calculation formula, the neutralization titer IC of the recombinant expressed antibody obtained in Example 1 against the F genotype wild strain is 50 As shown in Table 4, it is shown that the recombinantly expressed antibodies disclosed herein can recognize cells infected with the wild-type F genotype virus strain, neutralize the virus, and inhibit the spread of the virus between cells.

[0163] Table 4

[0164]

[0165] According to the above method and calculation formula, the recombinant expressed antibody obtained in Example 1 was subjected to neutralization experiments with the A genotype S79 vaccine strain (from Dalian Kexing) and the G genotype wild strain (isolated from the throat swab samples of children with clinical mumps infection), and the neutralization titer IC 50 As shown in Table 5, it is shown that the recombinantly expressed antibodies disclosed herein can cross-recognize cells infected with mumps viruses of various genotypes and play a role in neutralizing the viruses, and have a good broad-spectrum neutralization effect.

[0166] Table 5

[0167]

[0168] In summary, the present invention has successfully screened monoclonal antibodies with high binding efficacy and neutralizing activity for mumps virus, which can specifically identify mumps virus, have high neutralizing activity against F genotype mumps strains, and also have extensive cross-neutralizing activity with A genotype and G genotype, and have good application prospects for the detection, diagnosis, prevention and clinical treatment of mumps virus. The acquisition of high neutralizing efficacy antibodies for mumps virus is conducive to filling the current shortage of long-term protective power of mumps-containing vaccines, as well as causal treatment after mumps infection, alleviating symptoms and shortening the course of the disease, which has important clinical significance.

[0169] The above examples illustrate that the present disclosure can well screen mumps virus-type universal antibodies, and the antibodies have high antigenic targeting and low non-specificity. The acquisition of mumps virus universal antibodies is conducive to the efficient analysis of the immunogenicity level of multivalent mumps virus, which is of great significance for the non-clinical and clinical evaluation of multivalent vaccines.

[0170] The technical solution of the present disclosure is not limited to the above-mentioned specific embodiments, and all technical variations made according to the technical solution of the present disclosure fall within the protection scope of the present disclosure.

Claims

1. A method for preparing an antibody or an antigen-binding fragment thereof that targets and binds to mumps virus, the method comprising immunizing an animal with a whole virus of mumps virus to obtain the antibody or the antigen-binding fragment thereof.

2. The method according to claim 1, characterized in that The method further comprises screening the antibody or antigen-binding fragment thereof using an extracellular domain fragment containing a hemagglutinin-neuraminidase protein (HN protein) of mumps virus as a target protein; Preferably, the extracellular domain fragment of the HN protein has amino acids 106 to 582 corresponding to the HN protein of the F genotype strain of mumps virus; Preferably, the extracellular domain fragment of the HN protein has the amino acid sequence of SEQ ID NO: 39, or an amino acid sequence having at least 80% sequence identity thereto; Preferably, a secretory expression signal peptide is connected to the N-terminus of the extracellular domain fragment of the HN protein.

3. The method according to claim 1, characterized in that The whole virus of the mumps virus includes the whole virus of the F genotype mumps virus; and / or The animal comprises a mouse, a rat, a rabbit, a guinea pig, a sheep or a goat; and / or The method comprises preparing the antibody or antigen-binding fragment thereof by using a phage display method, a hybridoma antibody preparation method and / or a single B cell screening method; Preferably, the antibody is a monoclonal antibody.

4. The antibody or antigen-binding fragment thereof obtained by the method according to any one of claims 1 to 3.

5. An antibody or antigen-binding fragment thereof that targets and binds to mumps virus, the antibody or antigen-binding fragment thereof comprising: (1) The following three heavy chain variable region complementarity determining regions (HCDRs): HCDR1 having the amino acid sequence of HCDR1 contained in the heavy chain variable region as shown in any one of SEQ ID NO: 1, 3, 5, 7 or 10, or an amino acid sequence having one or more amino acids substituted, deleted or added compared to the amino acid sequence of HCDR1 contained in the heavy chain variable region; HCDR2 having the amino acid sequence of HCDR2 contained in the heavy chain variable region as shown in any one of SEQ ID NO: 1, 3, 5, 7 or 10, or an amino acid sequence having one or more amino acids substituted, deleted or added compared to the amino acid sequence of HCDR2 contained in the heavy chain variable region; HCDR3 having the amino acid sequence of HCDR3 contained in the heavy chain variable region as shown in any one of SEQ ID NO: 1, 3, 5, 7 or 10, or an amino acid sequence having one or more amino acids substituted, deleted or added compared to the amino acid sequence of HCDR3 contained in the heavy chain variable region; and / or (2) The following three light chain variable region complementarity determining regions (LCDR): LCDR1 having an amino acid sequence of LCDR1 contained in a light chain variable region as shown in any one of SEQ ID NOs: 2, 4, 6, 8, 9 or 11, or an amino acid sequence having one or more amino acids substituted, deleted or added compared to the amino acid sequence of LCDR1 contained in the light chain variable region; LCDR2 having an amino acid sequence of LCDR2 contained in a light chain variable region as shown in any one of SEQ ID NOs: 2, 4, 6, 8, 9 or 11, or an amino acid sequence having one or more amino acids substituted, deleted or added compared to the amino acid sequence of LCDR2 contained in the light chain variable region; LCDR3, which has the amino acid sequence of LCDR3 contained in the light chain variable region as shown in any one of SEQ ID NO: 2, 4, 6, 8, 9 or 11, or an amino acid sequence having one or several amino acids substituted, deleted or added compared to the amino acid sequence of LCDR3 contained in the light chain variable region.

6. The antibody or antigen-binding fragment thereof according to claim 5, characterized in that: The antibody or antigen-binding fragment thereof comprises: HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region as shown in SEQ ID NO:1, and LCDR1, LCDR2 and LCDR3 contained in the light chain variable region as shown in SEQ ID NO:2; or HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region as shown in SEQ ID NO:3, and LCDR1, LCDR2 and LCDR3 contained in the light chain variable region as shown in SEQ ID NO:4; or HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region as shown in SEQ ID NO:5, and LCDR1, LCDR2 and LCDR3 contained in the light chain variable region as shown in SEQ ID NO:6; or HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region as shown in SEQ ID NO:7, and LCDR1, LCDR2 and LCDR3 contained in the light chain variable region as shown in SEQ ID NO:8; or HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region as shown in SEQ ID NO:3, and LCDR1, LCDR2 and LCDR3 contained in the light chain variable region as shown in SEQ ID NO:9; or HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region as shown in SEQ ID NO: 10, and LCDR1, LCDR2 and LCDR3 contained in the light chain variable region as shown in SEQ ID NO: 11; Preferably, the HCDR1-3 and / or the LCDR1-3 are defined by the Kabat numbering system, the Chothia numbering system, the IMGT numbering system, the Martin numbering system, the Contact numbering system, the Honegger numbering system, the Gelfand numbering system or a combination thereof.

7. The antibody or antigen-binding fragment thereof according to claim 5, characterized in that: The heavy chain variable region of the antibody or antigen-binding fragment thereof comprises: HCDR1 as shown in any one of SEQ ID NO: 12, 22 or 33; HCDR2 as shown in any one of SEQ ID NO: 13, 18, 23 or 34; and / or HCDR3 as shown in any one of SEQ ID NO: 14, 24, 28 or 35; and / or The light chain variable region of the antibody or antigen-binding fragment thereof comprises: LCDR1 as shown in any one of SEQ ID NO: 15, 19, 25, 29, 30 or 36; and / or LCDR2 as shown in any one of SEQ ID NO: 16, 20, 26, 31 or 37; and / or LCDR3 as shown in any one of SEQ ID NO: 17, 21, 27, 32 or 38; Preferably, the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, respectively, and the light chain variable region of the antibody or antigen-binding fragment thereof comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, respectively; Preferably, the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 12, SEQ ID NO: 18 and SEQ ID NO: 14, respectively, and the light chain variable region of the antibody or antigen-binding fragment thereof comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively; Preferably, the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24, respectively, and the light chain variable region of the antibody or antigen-binding fragment thereof comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:27, respectively; Preferably, the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 28, respectively, and the light chain variable region of the antibody or antigen-binding fragment thereof comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 29, SEQ ID NO: 20 and SEQ ID NO: 21, respectively; Preferably, the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 12, SEQ ID NO: 18 and SEQ ID NO: 14, respectively, and the light chain variable region of the antibody or antigen-binding fragment thereof comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, respectively; Preferably, the heavy chain variable region of the antibody or its antigen-binding fragment comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:33, SEQ ID NO:34 and SEQ ID NO:35, respectively, and the light chain variable region of the antibody or its antigen-binding fragment comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38, respectively.

8. The antibody or antigen-binding fragment thereof according to claim 5, characterized in that: The antibody or antigen-binding fragment thereof comprises: A heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NO: 1, 3, 5, 7 or 10, an amino acid sequence having one or more amino acids substituted, deleted or added thereto, or an amino acid sequence having at least 80% sequence identity thereto; and / or A light chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 2, 4, 6, 8, 9 or 11, an amino acid sequence having one or more amino acids substituted, deleted or added thereto, or an amino acid sequence having at least 80% sequence identity thereto; Preferably, the antibody or antigen-binding fragment thereof comprises: The heavy chain variable region shown in SEQ ID NO: 1 and the light chain variable region shown in SEQ ID NO: 2; or The heavy chain variable region shown in SEQ ID NO:3 and the light chain variable region shown in SEQ ID NO:4; or The heavy chain variable region shown in SEQ ID NO:5 and the light chain variable region shown in SEQ ID NO:6; or The heavy chain variable region shown in SEQ ID NO:7 and the light chain variable region shown in SEQ ID NO:8; or The heavy chain variable region shown in SEQ ID NO:3 and the light chain variable region shown in SEQ ID NO:9; or The heavy chain variable region is shown as SEQ ID NO:10 and the light chain variable region is shown as SEQ ID NO:

11.

9. The antibody or antigen-binding fragment thereof according to claim 5, characterized in that: The antibody is of IgA, IgD, IgE, IgG or IgM type; Preferably, the antibody or antigen-binding fragment thereof is a scFv, Fab, Fab', (Fab')2, Fv fragment, dsFv, diabody, bispecific antibody and multispecific antibody; Preferably, the antibody or antigen-binding fragment thereof is a chimeric antibody or a humanized antibody. Preferably, the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region and / or a light chain constant region; More preferably, the heavy chain constant region is selected from IgG1, IgG2, IgG3 or IgG4 heavy chain constant region.

10. A chimeric antigen receptor comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain, wherein the extracellular antigen binding domain comprises an antibody or antigen binding fragment thereof prepared by the method of any one of claims 1 to 4, or an antibody or antigen binding fragment thereof according to any one of claims 5 to 9.

11. An isolated nucleic acid encoding an antibody or antigen-binding fragment thereof prepared by the method of any one of claims 1 to 4, or an antibody or antigen-binding fragment thereof according to any one of claims 5 to 9.

12. An expression vector comprising the isolated nucleic acid according to claim 11.

13. A host cell comprising the isolated nucleic acid according to claim 11, or the expression vector according to claim 12.

14. An antibody-drug conjugate comprising: an antibody or an antigen-binding fragment thereof prepared by the method of any one of claims 1 to 4, or an antibody or an antigen-binding fragment thereof according to any one of claims 5 to 9; and a ligand; Preferably, the ligand is selected from the group consisting of a radioisotope, a fluorescent group and a delivery vehicle.

15. A pharmaceutical composition comprising: an antibody or an antigen-binding fragment thereof prepared by the method of any one of claims 1 to 4, or an antibody or an antigen-binding fragment thereof according to any one of claims 5 to 9, or an antibody-drug conjugate according to claim 14; and a pharmaceutically acceptable carrier.

16. Use of the antibody or antigen-binding fragment thereof prepared by the method of any one of claims 1 to 4, or the antibody or antigen-binding fragment thereof according to any one of claims 5 to 9, or the isolated nucleic acid according to claim 11, or the antibody-drug conjugate according to claim 14, or the pharmaceutical composition according to claim 15 in the preparation of a medicament for preventing, treating and / or diagnosing a disease caused by mumps virus, or in detecting mumps virus or HN protein of mumps virus in vitro.

17. A kit for detecting mumps virus or HN protein of mumps virus in vitro, characterized in that: An antibody or antigen-binding fragment thereof prepared by the method of any one of claims 1 to 5, or an antibody or antigen-binding fragment thereof according to any one of claims 6 to 9, or an isolated nucleic acid according to claim 11; Preferably, the antibody or antigen-binding fragment thereof is labeled with a marker; More preferably, the label is selected from an enzyme, a chemiluminescent group and an isotopic group.

18. A method for preventing and / or treating mumps virus infection, comprising administering to a subject an effective amount of an antibody or an antigen-binding fragment thereof prepared by the method of any one of claims 1 to 5, an antibody or an antigen-binding fragment thereof according to any one of claims 6 to 9, a chimeric antigen receptor according to claim 10, an antibody-drug conjugate according to claim 14, or a pharmaceutical composition according to claim 15.

19. A method for detecting mumps virus or HN protein of mumps virus in a sample, the method comprising the step of detecting mumps virus or HN protein of mumps virus in a sample using the antibody or antigen-binding fragment thereof prepared by the method of any one of claims 1 to 5, or the antibody or antigen-binding fragment thereof of any one of claims 6 to 9, Preferably, the sample is whole blood, red blood cell concentrate, platelet concentrate, white blood cell concentrate, tissue, bone marrow aspirate, plasma, serum, cerebrospinal fluid, feces, urine, cultured cells, saliva, oral secretions and / or nasal secretions.

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